Laboratory science has undergone major advances in the last decade with increases in the speed and throughput of experiments and complexity of content (number of determinations per experiment). New technology has lead to dramatic increases in both the rate of performing assays and the rate of synthesis of new chemical compounds. The large volume of sequence determinations required to elucidate the human genome necessitated the development of high throughput equipment. Contemporary experimental molecular biology continues to drive the development of equipment for both high throughput and high content performance.
The need for high throughput equipment is driven by the requirements of the pharmaceutical industry. The drivers for this are the explosions in the rate of identification of drug targets brought about by genomics and proteomics research and the rate of synthesis of new chemical compounds through combinatorial chemistry approaches. The ability to test large numbers of candidate drug compounds contained in compound libraries against large numbers of drug targets has been a bottleneck for the pharmaceutical industry.
High content equipment performs many different determinations in a single experiment. For example DNA micro-arrays and protein chips have been developed to study an ensemble of genes or proteins in a cell in a single experiment as they are affected by a particular disease or treatment.
Whether it be for nucleic acid sequence determinations, single nucleotide polymorphism determinations or gene expression experiments in the field of genomics, for protein expression or protein function studies in the field of proteomics, or for testing of compounds in drug discovery, there continues to be a need for ever higher throughput and higher content analytical equipment.
It has become apparent to molecular biologists and drug development scientists that the increase in the rate of throughput of experiments to test potential pharmaceutical compounds has not yet resulted in a commensurate improvement in the rate of drug discovery. As scientists continue to uncover the complexity of cellular processes: the vastly larger complement of proteins in the proteome than the number only recently inferred from a one protein one gene model, the subtlety of interactions between proteins in signal transduction processes, and in the orchestration of gene control by the myriad of proteins controlling transcription, they have discovered that regulatory processes (and the disease states resulting from defects in these processes) depend on pathways that are the integration of multiple signals and stimuli. Cellular processes utilize concentration dependent signaling reactions, and interactions that are both time dependent and location specific within the cell. Of the modest numbers of compounds (relative to the almost limitless quantity of 1060 potential candidate compounds) being tested in current high throughput experimentation, those showing activity towards a particular target protein or nucleic acid reaction (enhancing or inhibiting a receptor/ligand binding interaction or enhancing or inhibiting an enzyme-substrate reaction for example) typically also affect many other reactions. The subtleties and vast complexities of biological processes reveal the limitations of simplistic single factor, equilibrium or steady-state in-vitro assays that have been used in the prior-art high throughput experimentation. To more closely imitate the complex in-vivo reactions, more complex multi-parameter in-vitro assay formats are being used, including high content assays utilizing living cells within the in-vitro assay reactors. Future strategies for scale-up of experimentation will necessitate both an increase in scale and an increase in the content of experimentation to levels that were heretofore not recognized, performed in devices that do not significantly sacrifice assay performance as they are scaled to high throughput and high content. Since the pharmaceutical industry's budgets allocated to these endeavors will not increase commensurately, it is clear that technologies are needed that will perform at an order of magnitude higher throughput and content than available with technology of the current art, and at an order of magnitude lower cost per data point without sacrificing the quality of the data relative to that obtained in low throughput assays.
The most widely adopted strategy to achieve high throughput or high content in analytical equipment is to perform a large number of assays in parallel. While there have been several quite different technologic approaches to scale-up of experiments through parallel processing as discussed below, almost all have two essential features in common. First, the apparatus of the parallel process approach comprises an array of micro-reactors generally arranged on a planar solid support. Second, the route to scale-up to high throughput is through miniaturization. Target molecules, such as fragments of DNA, RNA and proteins, either in solution or in living cells or drug candidate chemical compounds are often only available in minute quantities and they are expensive. The cost of reagents and samples is the dominant cost of an experiment in today's technology. Thus, with miniaturization as the route to scale-up the quantity of reagents and sample per assay and hence the cost per assay can also be significantly reduced.
One technologic approach to high throughput parallel experimentation on arrays has been to scale-up long-established small-scale parallel experiments such as those performed on micro-plates. Planar arrays of micro-reactors in wells on micro-plates are being scaled up to high throughput by increasing the number of wells on the plate, thereby also decreasing the volume of each well (for recent examples see U.S Pat. No. 6,229,603 B1). High throughput equipment of the current art routinely employs standard sized 12.8 cm×8.6 cm plates with 96 and 384 wells. Plates with 1536 wells are now being introduced and ultra high throughput apparatus consisting of up to 9600 wells on a single standard size plate are also known. The industry would like to move to 9600 wells per plate or more. Reaction volumes in today's micro-plate technology are 1 microliter or more, but there is a need to develop devices requiring much smaller reaction volumes particularly for those applications where very little sample is available, or the reagents are very expensive.
Each well of the micro-plate array supports a discrete micro reaction. In the current art micro-liter quantities of sample are introduced into each well, along with other reagents undergoing the chemical reaction. A detector for monitoring the chemical reaction probes each well. Optical detection such as fluorimetry is a preferred approach. In a typical use of this high throughput device, aliquots of different chemical compounds are transferred from a compound library plate into the assay plate by a parallel fluid-dispensing manifold. The transfer of sample and other assay reagents is by robot-controlled fluid handling means including an array of micro-pipettors, capillary tubes, pumps and the like. Both homogeneous and heterogeneous reactions are performed in planar arrays of wells. Homogeneous enzyme-substrate reactions, and the effect of candidate drug compounds on them, can be monitored by change of fluorescence intensity using a fluorogenic substrate. Homogeneous, solution phase receptor/ligand binding reactions, and the effect of candidate drug compounds on them, can be monitored by one of a number of fluorescence based techniques the most popular being fluorescence polarization (for example U.S. Pat. Nos. 5,641,633 and 5,756,292 for fluorescence polarization assays for nucleic acids). In heterogeneous reactions a heterogeneous binding reaction takes place when one of the reactants is attached to a solid surface. Reagents or sample can be immobilized on the wells' surfaces or they can be immobilized on the surface of beads introduced into the reaction wells (for example, U.S. Pat. No. 6,210,891 B1 describing a nucleic acid primer extension reaction on a bead immobilized DNA sample).
At low levels of integration, the micro-plate reactor-array can accomplish complex experimental formats such as those with numerous reagent additions, timed reactions, washes, bead separations and the like. But these complex reaction formats are difficult and expensive to miniaturize and automate to highly parallel operation, because the fluidic input and output devices supplying chemicals to or removing chemicals from the micro-reactor wells (the fluidic i/o) become too complex. Consequently, significant resources are being applied to the extension of the use of simple and rapid equilibrium bimolecular homogeneous reaction formats that can be more easily automated to highly parallel operation and low reaction volume. Because of the time delay in delivering reagents to high density array plates, time-transient measurements are not possible. Multiple dosing of each well also has not been possible at high density. Instead dose response curves are generated from multiple wells operating the same reaction at different concentration levels of a reactant.
Workers in the field of micro-arrays have taken a different approach to parallel experiments. Micro-arrays are devices consisting of dry reagents immobilized in arrays on non-porous planar substrates. Micro-arrays perform high content assays: many heterogeneous receptor/ligand binding micro-reactions in parallel on a single sample. In these devices, the planar support surface, often a glass slide, a glass plate or a silicon wafer, consists of an array of reaction micro-locations, each location containing a different chemical compound attached to the surface of the planar substrate. In the most common form of this technology, a fluorescence reader or scanner detects the chemical reaction taking place in each micro-location. In use, the array is immersed in a bath containing sample for analysis as well as other chemicals for reaction at the planar micro-locations. Only heterogeneous reactions are performed in devices of this type. Workers in the genomics field have developed micro-arrayed nucleic acids (cDNA and oligonucleotides) attached to planar surface in which case the devices are also called gene-chips or printed DNA arrays. A series of recent review articles on this topic can be found in Nature Genetics Supplement, vol. 21(1), January 1999. Each micro-location contains a nucleic acid with a specific sequence of bases attached to the surface. Typically the base sequence of each micro-location is different. In use, the nucleic acid micro-array is exposed to a test fluid containing polynucleic acids (DNA, RNA or pDNA) to be assayed. Polynucleic acids in the test fluid have been previously labeled by attachment of a reporter molecule such as a fluorescent tag. There is a strong binding reaction between polynucleic acids in the test fluid having a base sequence complimentary to the base sequence of the nucleic acid attached to the micro-location of the array. After the binding step, a washing step removes unbound polynucleic acids from the micro-locations. The fluorescence scanner then reads the micro-array. A binding reaction at a micro-location is detected by fluorescence at that site. Nucleic acid hybridization micro-arrays have been used to perform sequencing experiments (U.S. Pat. Nos. 5,202,231 and 5,695,940) and to determine the presence of specific nucleic acid sequence variants such as single nucleotide polymorphisms (U.S. Pat. No. 5,837,832). The widest use of micro-arrays however has been in the field of gene expression (see for example chapter 7 of the book “Microarray Biochip Technology” ed. Mark Schena, Eaton Publishing 2000).
There are several variations of the nucleic acid micro-array including arrays of oligonucleotides attached to a surface (U.S Pat. Nos. 5,445,934 5,744,305 and 5,700,637), either fabricated in-situ using photolithographic masking processes (U.S. Pat. Nos. 5,405,783 and 5,489,678) and ink-jet printing (see for example T. R. Hughes et al. Nature Biotechnology. vol. 19, p342–347, 2001) or fabricated off-chip then applied to the planar substrate by an array spotter (see for example U.S. Pat. No. 5,807,522). Another variant is the cDNA array also fabricated by spotting. Genomics researchers have proposed extending the scope of the micro-array beyond nucleic acid hybridizations to include for example PCR on micro-arrays (U.S. Pat. No. 6,248,521) and primer extensions on micro-arrays (U.S. Pat. Nos. 5,547,839 and 6,210,891).
One aspect of the micro-array that has been responsible for its success is the ability to perform high content (many different receptor/ligand binding experiments: nucleic acid hybridization or protein binding) in a single batch process using very little sample and reagent. In nucleic acid hybridization for example, using a micro-array with 20,000 reaction sites on a glass slide immersed in about 1 mililiter of sample, the reaction volume of each hybridization taking place over a 100 micrometer diameter spot containing picomole quantities of attached oligonucleotide is of the order of about 50 nano-liters. Another aspect of the success of the micro-array is the inherent simplicity of the procedure. It is well known, however, that nucleic acid hybridization thermodynamics and kinetics are sequence dependent, so that for a single experimental condition the amount of hybridization occurring at two sites for which there is a positive sequence match might be quite different. For this and other reasons, the simple hybridization micro-array of the current art is not a quantitative device. Differential or comparison hybridization methods have been developed in light of this limitation (see for example chapter 7 of the book “Microarray Biochip Technology” ed. Mark Schena, Eaton Publishing 2000). In a typical differential gene expression experiment, two samples of cDNA are co-hybridized onto an array. cDNA prepared from RNA extracted from cells under study is labeled with fluorescent dye cyanine-3 (or cyanine-5). cDNA prepared from RNA extracted from control cells is labeled with cyanine-5 (or cyanine-3). The relative amount of hybridization, as measured at the two different wavelengths of the cyanine-3 and cyanine-5 fluorescence, indicates the level of expression of a particular gene in the study cells relative to the control. An approach used to control hybridization and presumably result in better quantitation is described in U.S. Pat. Nos. 5,632,957, 5,653,939 and 6,017,696 where micro-arrays with site-specific electronic addressing are taught, claiming site specific control of hybridization stringency conditions via the voltage applied to an electrode immediately under the hybridization site. Another problem with current DNA micro-array technology is the difficulty of measuring low concentrations. In the gene expression experiment, mRNAs with low abundance (one transcript per cell or less) cannot easily be determined particularly when using RNA collected from only a small number of cells. However, low concentration signaling proteins translated from low abundance mRNAs are often the most interesting to study. An enzyme amplification technique using tyramide signal amplification has been adapted to gene expression arrays to improve the detection limit by 10 to 50 fold (see for example Adler et al. in chapter 10 of Microarray Biochip Technology ed. Mark Schena).
Protein arrays using the same design principles as nucleic acid arrays have been disclosed for clinical diagnostic applications (U.S. Pat. No. 5,432,099). More recently protein micro-arrays have been developed to study protein-protein interactions in high throughput molecular biology applications (MacBeath et al. Science, 289 (5485), pp 1760–1763, 2000).
Unlike nucleic acids in the DNA micro-array experiments, which are assayed as free molecules and do not complex, proteins in a sample of cellular extract are not present just as single discrete molecules but rather they are bound in often numerous multi-molecular protein complexes. In the case of cellular protein binding, the kinetics and thermodynamics of binding reactions are particular to a protein and its binding partner. Binding constants (K) vary widely (106<K<1013 L/mole). Binding constants of proteins to capture molecules on an array surface also will be widely varying. Binding constants of cellular proteins either one to another in complex formation or to capture molecules in an array (both free and complexed proteins being captured), are dependent on the reaction environment: temperature, pH, ionic strength, hydrophilic versus lipophilic environment, concentration of specific ions and dissolved oxygen, cofactors and the like. Also, the relative amounts of free and complexed protein will depend on the concentration and therefore will be strongly affected by the amount of dilution of the cellular extract used in an experiment.
As in a nucleic acid array, in a protein chip there are many different types of capture molecules arrayed on a planar substrate which is immersed in a sample of cellular extract. At a particular capture location, a capture molecule has been designed to capture a single particular protein molecule type (call it A) with good specificity over other proteins in the sample (one part in 106 is often cited as a benchmark for specificity). The protein molecule A will be captured at that site along with multi-molecular complexes containing A (which contain other proteins including a protein B). Thus there will be many non-A proteins captured at the A capture site including protein B. At the capture site designed to specifically capture protein B there will be free B and B complexes including some protein A. Accordingly the specificity of a single capture site to its binding partner is lost. Such a device will be rendered useless unless the various components of the signal could be de-convoluted by ab initio knowledge of all of the binding constants involved. For a large multi-component array this is not practical.
Accordingly, the simple protein array immersed in a single batch of sample should not be expected to deliver quantitative data. Nor is the data from this in-vitro experimental format likely to be an accurate model of the in-vivo interactions.
Thus, a general limitation of the high content nucleic acid and protein micro-array of the current art is that they can only perform simple bimolecular heterogeneous binding reaction formats.
Yet another approach to parallel experimentation in planar arrays has been taken by the lab-on-a-chip developers. The micro-reactors of this technology comprise micro-channels and cavities formed by etching or laser ablation of material from the surface of a planar glass substrate (U.S. Pat. No. 5,180,480) or polymer substrate (U.S. Pat. No. 5,750,015). The planar substrate with formed channels and cavities is capped with an insulating cover assembly. The capped channels and cavities now form capillary conduits and chambers collectively known in the art as micro-fluidics. When there is an opening in the cap over a chamber, it becomes a well for sample and reagent introduction. Aqueous sample and reagents are dispensed into the wells using a fluid-handling manifold in much the same way as in the micro-plate technology. The dispensed fluids then fill the empty capillary conduits of the device. In many micro-fluidic methods of the prior art, pumping is by electro-kinetic propulsion in which case an electrode manifold is then brought into contact with the aqueous solutions in the wells to provide the power to electro-kinetically pump fluids from wells through the capillary conduits. In the micro-fluidic array, each micro-location of the array constitutes a micro-fluidic reactor consisting of channels and wells. In the current art, the level of parallel processing in the lab-chip array is low compared to micro-plate technology, but the technology is also amenable to automated high speed serial experimentation, so that high throughput can obtained by a combination of serial and parallel operations. In the current art, the sample volume of commercial lab-chips is about 0.1 micro-liters per experiment. Lab-on-a-chip developers have disclosed a number of different capabilities of their micro-fluidic devices, including high throughput screening of candidate drug compounds (U.S. Pat. No. 6,150,180), macromolecule separations (U.S. Pat. No. 4,908,112), nucleic acid separations (for example Woolley et al. Proc. Natl. Acad. Sci. USA Vol. 91, pp11348–11352, 1994), polymerase chain reactions (U.S. Pat. No. 6,235,471 B1) and Sanger sequencing by dideoxy chain termination and sizing by capillary electrophoresis (U.S. Pat. No. 5,661,028). U.S. Pat. No. 6,103,479 discloses an array of micro-locations with different cell binding sites and bound cells on a planar surface mated with a micro-fluidic planar substrate with etched cavities and channels.
Although complex fluid-handling capability has been demonstrated within the etched channel structures, the lab-chip devices of this art are still only lab glassware on a chip. Conventional lab-on-a-chip devices employing electro-kinetic pumping cannot be easily adapted to assay formats incorporating on-board reagents, and the supply of chemicals and reagents from off-chip sources remains a significant problem, as it is in the micro-plate devices that support complex reaction formats. Thus, here too the ability to scale multi-component complex reaction formats to small volume and highly parallel operation is limited by the ability to provide the fluidic i/o to the lab chip. One developer of high throughput screening instruments has adapted the lab-on-a-chip device to sample small volume fluid aliquots from a micro-plate. In this device a lab-chip acquires in a serial manner sub micro-liter quantities of samples for reaction from the wells of a micro-plate using an electro-pipettor (U.S. Pat. Nos. 5,942,443 and 6,235,471). The lab-chip and integral electro-pipettor step over the micro-plate sampling each well in turn. To achieve high throughput, samples are rapidly run in the lab-chip in a serial reaction format. However, this approach is limited because it only scales to high throughput when each assay can be run rapidly.
Yet another approach to parallel experimentation is the collection of methods known as solid-phase reaction formats. In these methods reactions are performed on planar slabs of porous or gelatinous materials. Devices of this art include nucleic acid arrays on porous substrates and gels such as those used in traditional blotting techniques, multi-lane gel slabs for parallel electrophoresis separations can be classified as solid phase reactions (see for example U.S. Pat. No. 5,993,634) and arrays spotted onto reagent impregnated planar gel slabs in continuous format high throughput screening technology (U.S. Pat. No. 5,976,813). In the continuous format approach, sample is spotted onto a planar porous slab that is laminated with one or more other planar slabs containing reaction reagents. At the time of the assay, sample and reagents intermix by diffusion between slabs. Using this approach, the continuous format devices avoid the fluidic i/o complexity of the other array technologies. However, the spot separation is relatively large (several milimeters) because individual reaction micro-locations must be sufficiently well separated to avoid mixing between reaction chemicals of adjacent micro-locations when they diffuse along the planar slab. Sample volumes are large, being in the 1 to 10 micro-liter range. Reagent volumes are much larger because the reagent containing slabs have large unused inter-spot areas.
In summary, high throughput micro-reactor arrays of the prior-art are limited in one of several ways. Micro-plate wells, even highly parallel 1536 well plates, at the current state of the art still require relatively large micro-liter volumes of sample and reagents. The cost per assay is thus still much too high. These devices are effective for performing single step bimolecular homogeneous reaction and can be further scaled to more parallel operation and somewhat lower volume, but they will not easily achieve the micro-reactor densities or nano-liter reaction volume achievable on micro-arrays. Furthermore, multi-component reaction formats such as those requiring timed delivery of one or multiple sample aliquots and/or multiple reagents, wash steps or purifications and separation steps are too complicated for scale-up in micro-plate technology. Lab-on-a-chip devices which operate on sub-micro-liter reaction volumes are similarly limited in ability to scale-up to highly parallel operation because of fluidic i/o complexity. Lab-on-a-chip devices operating in serial reaction format are not easily adaptable to heterogeneous binding assays and they are limited to assays with short reaction times. Continuous format gel-slab reactors use micro-liter sample volumes. Only micro-arrays of the current art exhibit highly parallel operation and have been miniaturized to tens of nano-liters reaction volumes. But they are limited in the scope of their utility, generally performing only single step heterogeneous binding reactions. Micro-arrays of the current art are further limited because the parallel reactions are run as a single batched experiment under identical conditions for all micro-locations of the array. Furthermore, micro-arrays of the current art are not very suitable for protein expression studies.
Thus there is a need for a technology that will provide complex reaction formats in high-density arrays with nano-liter reaction volumes. As a route to achieve this there is a need for a technology that will provide miniaturized, highly parallel reaction capability with simple, cost-effective fluidic i/o. To simply state the problem with prior-art technology: it has not been possible to introduce sub pico-mole quantities of chemicals dissolved in sub nano-liter quantities of solution to a micro-location of an array in real time.